Dataset Information

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.

ABSTRACT: Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.

SUBMITTER: Fu J

PROVIDER: S-EPMC2553598 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.

Fu Jun J Wenzel Silke C SC Perlova Olena O Wang Junping J Gross Frank F Tang Zhiru Z Yin Yulong Y Stewart A Francis AF Müller Rolf R Zhang Youming Y

Nucleic acids research 20080813 17

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of ...[more]

PMID: 18701643

Similar Datasets

Project description:BACKGROUND:Heterologous expression of secondary metabolite gene clusters is used to achieve increased production of desired compounds, activate cryptic gene clusters, manipulate clusters from genetically unamenable strains, obtain natural products from uncultivable species, create new unnatural pathways, etc. Several Streptomyces species are genetically engineered for use as hosts for heterologous expression of gene clusters. S. lividans TK24 is one of the most studied and genetically tractable actinobacteria, which remain untapped. It was therefore important to generate S. lividans chassis strains with clean metabolic backgrounds. RESULTS:In this study, we generated a set of S. lividans chassis strains by deleting endogenous gene clusters and introducing additional φC31 attB loci for site-specific integration of foreign DNA. In addition to the simplified metabolic background, the engineered S. lividans strains had better growth characteristics than the parental strain in liquid production medium. The utility of the developed strains was validated by expressing four secondary metabolite gene clusters responsible for the production of different classes of natural products. Engineered strains were found to be superior to the parental strain in production of heterologous natural products. Furthermore, S. lividans-based strains were better producers of amino acid-based natural products than other tested common hosts. Expression of a Streptomyces albus subsp. chlorinus NRRL B-24108 genomic library in the modified S. lividans ΔYA9 and S. albus Del14 strains resulted in the production of 7 potentially new compounds, only one of which was produced in both strains. CONCLUSION:The constructed S. lividans-based strains are a great complement to the panel of heterologous hosts for actinobacterial secondary metabolite gene expression. The expansion of the number of such engineered strains will contribute to an increased success rate in isolation of new natural products originating from the expression of genomic and metagenomic libraries, thus raising the chance to obtain novel biologically active compounds.

Project description:Recent reviews have reinforced sponge-associated bacteria as a valuable source of structurally diverse secondary metabolites with potent biological properties, which makes these microbial communities promising sources of new drug candidates. However, the overall diversity of secondary metabolite biosynthetic potential present in bacteria is difficult to access due to the fact that the majority of bacteria are not readily cultured in the laboratory. Thus, use of cultivation-independent approaches may allow accessing "silent" and "cryptic" secondary metabolite biosynthetic gene clusters present in bacteria that cannot yet be cultured. In the present study, we investigated the diversity of secondary metabolite biosynthetic gene clusters (BGCs) in metagenomes of bacterial communities associated with three sponge species: Clathria reinwardti, Rhabdastrella globostellata, and Spheciospongia sp. The results reveal that the three metagenomes contain a high number of predicted BGCs, ranging from 282 to 463 BGCs per metagenome. The types of BGCs were diverse and represented 12 different cluster types. Clusters predicted to encode fatty acid synthases and polyketide synthases (PKS) were the most dominant BGC types, followed by clusters encoding synthesis of terpenes and bacteriocins. Based on BGC sequence similarity analysis, 363 gene cluster families (GCFs) were identified. Interestingly, no GCFs were assigned to pathways responsible for the production of known compounds, implying that the clusters detected might be responsible for production of several novel compounds. The KS gene sequences from PKS clusters were used to predict the taxonomic origin of the clusters involved. The KS sequences were related to 12 bacterial phyla with Actinobacteria, Proteobacteria, and Firmicutes as the most predominant. At the genus level, the KSs were most related to those found in the genera Mycolicibacterium, Mycobacterium, Burkholderia, and Streptomyces. Phylogenetic analysis of KS sequences resulted in detection of two known 'sponge-specific' BGCs, i.e., SupA and SwfA, as well as a new 'sponge-specific' cluster related to fatty acid synthesis in the phylum Candidatus Poribacteria and composed only by KS sequences of the three sponge-associated bacterial communities assessed here.

Dataset Information

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.

Publications

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets